Generation and characterization in this laboratory of of the activation antigen-specific mAb GL1 led to identification of the mouse B7-2 (CD86) costimulatory molecule, which has predominant functional costimulatory role both in vivo and in vitro. Anti-B7-2 mAb inhibited accessory cell-dependent responses of T cells in vitro and in vivo, indicating that B7-2 is a functional costimulatory molecule for T cell-dependent (TD) responses. Expression of B7-2 costimulatory molecules during in vivo T-dependent B cell antibody responses correlated with somatic hypermutation and affinity maturation, and in in vivo treatment with anti B7-2 inhibited TD responses as well as memory formation and somatic mutation. The use of mice rendered deficient in both B7-1 and B7-2 has further characterized the functional requirement for B7 costimulation in antibody responses in vivo. Experiments have assessed the requirement for both B7 and CD40 costimulation in in vivo T cell-dependent (TD) B cell activation, using mixed radiation bone marrow chimeras in which B7-deficient or CD40-deficient and wild type B cells co-exist. These studies have demonstrated that, although both costimulatory pathways are critical for TD responses, distinct mechanisms underlie the requirements for B7 and CD40. The requirement for CD40 is cell autonomous in the sense that CD40 must be expressed on a B cell in order for that B cell to respond to TD activation in vivo. In contrast, B7-deficient B cells are capable of efficient response if a B7-intact environment provides necessary exogenous factors. It has further been shown in mixed chimeric experiments that B7 expression by bone marrow-derived non-lymphoid cells is sufficient in these responses, and that B7 need not be expressed on any B or T cell in the generation of TD B cell responses. Transgenic mice expressing cytoplasmic deletion mutants of B7 are being generated to assess the role of functions including signal transduction through B7. T cell dependent (TD) B cell activation was analyzed in the antibody responses of mice defective in the ataxia telangiectasia (AT)mutated (Atm) gene. These mice were found to have a profound defect in vivo in Ig class switching during TD antibody responses, with most extensive defects observed in IgE and IgA response, paralleling the clinical AT syndrome. In vitro studies have identified a defect intrinsic to B lymphocytes in the ability to Ig switch in response to activation with cytokines plus LPS or anti-CD40. Cellular analysis has identified a defect in the process of Ig class switch recombination in Atm-deficient B cells, consistent with a role of the Atm product in the process of DNA break/repair involved in this recombination process. Molecular analysis of in vitro responses indicated that WT and Atm-/-B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm-/- B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm-/- B cells occurs at the level of genomic DNA recombination as measured by digestion-circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the m-g1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.